Automated microscopic cell analysis

ABSTRACT

This disclosure describes single-use test cartridges, cell analyzer apparatus, and methods for automatically performing microscopic cell analysis tasks, such as counting blood cells in biological samples. A small unmeasured quantity of a biological sample such as whole blood is placed in the disposable test cartridge which is then inserted into the cell analyzer. The analyzer isolates a precise volume of the biological sample, mixes it with self-contained reagents and transfers the entire volume to an imaging chamber. The geometry of the imaging chamber is chosen to maintain the uniformity of the mixture, and to prevent cells from crowding or clumping, when it is transferred into the imaging chamber. Images of essentially all of the cellular components within the imaging chamber are analyzed to obtain counts per unit volume. The devices, apparatus and methods described may be used to analyze a small quantity of whole blood to obtain counts per unit volume of red blood cells, white blood cells, including sub-groups of white cells, platelets and measurements related to these bodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/017,498, filed Feb. 5, 2016, which is a continuation in part of U.S.application Ser. No. 14/947,971, filed Nov. 20, 2015, which claimspriority to U.S. provisional application Nos. 62/138,359, filed Mar. 25,2015, 62/113,360 filed Feb. 6, 2015, and 62/084,760, filed Nov. 26,2014, which are all herein incorporated by reference.

FIELD OF THE INVENTION

This invention pertains to analyzers and methods for automaticallyperforming microscopic cell analysis tasks, such as counting blood cellsin biological samples. More specifically, the present disclosure relatesto single use devices, apparatus and methods used to count red bloodcells, white blood cells and platelets, and measurements related tothese bodies.

BACKGROUND OF THE INVENTION

There are a variety of methods for enumerating particles, such as cellsin a biological sample. Determining the number of cells per unit volumein a sample provides the physician important diagnostic information. Themost elementary method of counting cells consists of introducing adiluted biological sample into a hemocytometer and examining it with amicroscope. A hemocytometer is a device with an optically clear chamberhaving a known depth, typically 100 microns, and ruled markings todefine a unit volume, typically 0.01 μL. A uniform mixture of dilutedwhole blood, for example, may be introduced into the hemocytometer bycapillary action to form a monolayer. Using a microscope to visualizethe diluted sample, cells of different types can be counted manually ina limited number of marked areas. Counts are aggregated to compute thenumber of cells per unit volume. This manual method is time consuming,tedious, and requires a skilled technician to operate the microscope andto recognize the various types of cells, and is prone to error. Itsaccuracy is limited by the number of cells counted and the homogeneityof the monolayer formed by introduction of the diluted sample.

Consequently, automated methods, such as impedancemetry (Coulterprinciple U.S. Pat. No. 2,656,508) and flow cytometry, have beendeveloped for rapid counting, sizing, and classification of a relativelylarge number of cells for diagnostic tests such as the Complete BloodCount (CBC) or CBC with a five part differential. These automatedmethods also have shortcomings. The analyzers are relatively large andexpensive and require skilled operators for their use and maintenance.Such analyzers are typically available only in centralized laboratories.Blood samples are collected in special containers having ananticoagulant to keep the blood from clotting while being transported tothe lab. This process adds costs and risk of erroneous results fromtransport, handling, labeling and transcription, as well as a time delayin obtaining the results.

The CBC generally includes measures of white blood cells (leukocytes)per unit volume (WBC), red blood cells (erythrocytes) per unit volume(RBC), platelets (thrombocytes) per unit volume (PLT), hematocrit (HCT)or packed cell volume (PCV), hemoglobin (HGB), and measurements relatedto red cells including mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), mean corpuscular hemoglobin content (MCHC), and redcell distribution width (RDW). A diagnostic test referred to as a “CBCwith differential”, also known as a “CBC with five part diff”, will alsoinclude Neutrophil granulocytes (NEU), Lymphocytes (LYM), Monocytes(MON), Eosinophil granulocytes (EO) and Basophil granulocytes (BASO) perunit volume or as a percentage of the white blood cells (WBC). The CBCwith differential also may include counts of Immature Cells (IC),nucleated Red Blood Cells (nRBC), and Reticulocytes (RETIC) per unitvolume of the blood sample.

The CBC provides a panel of blood cell measurements that can be used todiagnose a wide variety of abnormal conditions, such as anemia orinfection, or to monitor a patient's treatment, such as chemotherapy.Because of its usefulness, the CBC analysis is one of the most commonlyperformed diagnostic tests in medicine, but patients typically wait aday or more for results. If microscopic cell analysis could be performedin a portable, easy-to-use, analyzer close to the patient, results couldhave a more immediate impact in improving patient care. A simple systemable to provide the CBC in the physician's office, or at bedside, or inthe Critical Care Unit (CCU) or Intensive Care Unit (ICU), within a fewminutes and using a drop of blood from a finger-stick, could haveenormous impact on the delivery and affordability of health care.

Recent patent documents have described simpler devices than centralizedlab hematology analyzers for performing cell analysis of blood samples.In U.S. Pat. No. 7,771,658, issued to Larsen, the applicant, providestechnology for performing a flow-cell analysis of blood cells in asingle use disposable cartridge. Larsen describes means for taking anexact amount of blood sample, diluting the amount of blood with aprecise volume of diluent, and mixing the blood with the diluent toobtain a homogeneous solution. Larsen utilizes a single use cartridge toflow a measured amount of the mixture of sample and diluent through anorifice at a rate of several thousand particles per second, and counts,sizes, and classifies the particles for analysis in accordance with theCoulter principle. Because Larsen's disclosure is directed to theanalysis of a small sample of whole blood, errors in metering variousvolumes or in the mixing or sampling steps can significantly impact theaccuracy of the results.

PCT Patent Number WO 2014099629 issued to Ozcan et al. describes asystem for analyzing a blood sample with a mobile electronic devicehaving a camera. The sample preparation process for each test requiresaccurate measurement of 10 μL of whole blood to be mixed with 85 μLphosphate buffered saline and 5 μL of nucleic acid stain. Ten (10) μL ofthis diluted mixed sample are then loaded into a cell counting chamberwith precise channel height of 100 μm and are imaged by a digitalcamera. A separate cell counting chamber is needed for analysis of redcells, white cells, and hemoglobin, and each test should be performedseparately. Accuracy of the final result is not only dependent on theaccurate measurements of the various sample preparation steps, includingthe precise metering of the sample and diluent, but also on the precisefabrication of the counting chambers. Maintaining uniformity andconsistency of the 100 μm dimension of the channel height in adisposable cartridge is difficult to achieve in a low cost device.

U.S. Pat. No. 8,837,803 to Wang et al. describes a method fordetermining cell volume of red blood cells, which seeks to avoid theerrors associated with diluting, mixing, and sampling, by analyzing asample of substantially undiluted whole blood. In theory this approachhas appeal, but the handling of undiluted whole blood is challenging.The cells are so numerous (for example 5,000,000 red cells in 1 μL) thattheir distribution can be impacted by contact with the surfaces of adisposable cartridge. Additionally, in order to image cells in thisovercrowded environment, the imaging chamber should have a depth of onlya few microns to prevent the overlapping of cells, and it should befabricated accurately, because it determines the volume of the bloodsample being analyzed.

Therefore a compact, accurate method of performing microscopic cellanalysis using digital camera imaging of a diluted sample, which doesnot require accurate measurement of the diluent volume, or requireaccurate or precise dimensions of an imaging chamber, is highlydesirable.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide an apparatus thatcan provide counts of cells or other particles of a biological sampleper unit volume without requiring an operator having specializedknowledge or specialized skills. Another objective of the presentinvention is to provide an easy-to-use apparatus that can perform all ofthe measurements of the CBC in a few minutes. Another objective of thepresent invention is to provide a method for determining theconcentration of particles in a diluted biological sample that does notrequire accurate measurement of a volume of diluent or reagent mixedwith the sample. Another objective of the present invention is toprovide ready-to-use reagents for use in performing the microscopicanalysis of a biological sample that do not require preparation, mixingor measurement by the user. Another object of the invention is to createa substantially homogenous monolayer of cells of a diluted sample in asingle use disposable device and to perform a CBC analysis in a fewminutes. Another objective is to provide a sample collection device thatcan easily be operated by a user to obtain a biological sample foranalysis. Yet another objective is to provide all of the fluidiccomponents of an apparatus to perform a CBC in a single-use device, soas to prevent cross contamination between samples and to avoid the needfor cleaning and washing of fluidic components. Another objective of thepresent invention is to provide internal monitoring of the criticalprocess steps of counting cells, so that a potentially erroneous resultcan be flagged. Still another objective is to provide a method ofperforming the CBC immediately after collecting the sample, and toeliminate transport of the sample to a laboratory or storage of thesample. Some or all of these and/or other objectives or advantages maybe accomplished by embodiments of the invention as provided for in theappended claims.

The disclosure provides improved methods, systems, and devices forperforming counts and measurements of particles in a biological sample.Aspects of the disclosure are directed to determining the concentrationof one or more types of particles and providing the result as the numberof such particles per unit volume of the biological sample in a fewminutes. The particles may be any mass suspended in a liquid that can berecognized by optical inspection using an automated microscope and imageanalysis techniques well known in the art. Examples of particlesinclude, but are not limited to, blood cells, sperm, bacteria, spores,and inorganic particles.

While embodiments of the present invention can be used in manyapplications to count particles suspended in a liquid, the presentdisclosure will demonstrate benefits of the invention in performing theComplete Blood Count (CBC) or CBC with differential on human bloodsamples as defined above. The present disclosure describes a single-usetest cartridge for use with an apparatus that includes an automatedmicroscope for analyzing cells in a biological sample. The testcartridge is used to collect a biological sample. For example the usercan prick the finger of a patient and obtain a whole blood sample andcollect the resulting drop of blood in the test cartridge. The user canaccomplish this by holding the test cartridge beneath the hanging dropfrom the patient's finger and then bringing it closer until the dropcontacts an input port or sample cup on the test cartridge. In analternate embodiment, the user may collect a blood sample intravenouslyand transfer it to the test cartridge using a capillary tube or plasticbulb pipette. In one example the single-use test cartridge includes aninput port adapted to accept a variety of sources of biological samplesincluding a direct sample, capillary tube, or transfer pipette. Inanother example, the test cartridge could draw a sample into it bycapillary action. The test cartridge may also include a closure that theuser applies to cover the input port after the sample has been collectedinto the test cartridge. The closure may be part of the test cartridgeor a separate device. The user may apply the closure manually or theapparatus may automatically move the closure to cover the input portwhen the test cartridge is docked to the apparatus. The closure providesa physical barrier to avoid subsequent contact with any excess sample onthe surface of the test cartridge. The closure has a vent to provide anair path to the input port to allow the liquid sample to move within thetest cartridge, as will be described below.

Within the test cartridge, the input port is connected by an inputchannel to a metering chamber capable of precisely separating a smallvolume of the sample from the unmeasured sample. As an example, thecollected sample volume may be 5-20 μL, of which the metering chamberseparates out or isolates 0.1-5 μL for measurement. If a finger sticksample is utilized, a sample volume less than 10 μL will minimize therisk of hemolysis and the risk of dilution by interstitial fluids. Themetering chamber can be a section of a fluid channel, a cavity, or apass-through conduit within a valve, or another volumetric shape thatcan be reproducibly fabricated to contain a predetermined volume of thebiological sample in the range of 0.1 to 5 μL. Injection molding,compression molding, etching or other processes known to those skilledin the making of lab-on-a-chip or microfluidic devices can be utilizedto manufacture the metering chamber.

In one example, the metering chamber is combined with a rotary valvestructure by molding a pass-through conduit in the cylindrical stem ofthe rotary valve. As an example a pass-through conduit having acylindrical cross section with 0.5 mm diameter and a length of 5 mm, hasan internal volume of approximately 1 μL. The pass-through conduit isinitially filled with the biological sample by providing a fluidcommunication path between the input port and input channel, thepass-through conduit, and a vacuum channel. A vacuum applied to thevacuum channel pulls the sample into the pass-through conduit.Alternatively the biological sample can be pulled into the pass-throughconduit by capillary attraction. Once the biological sample hascompletely filled the pass-through conduit, the cylindrical stem of therotary valve is rotated until the pass-through conduit is no longerconnected to the input channel or the vacuum channel, thus separating orisolating the sample volume contained in the pass-through conduit.Alternatively, a rotary face seal valve, a slide valve or a fixedgeometry in the test cartridge could be used to isolate a small volumeof the sample.

Further, elements of the test cartridge include a prepackaged liquidreagent or alternatively, a chamber for storing a liquid reagent,together with a mixing chamber, an imaging chamber, and fluid channelsthrough which the sample and liquid reagent may be moved. In oneexample, after the biological sample has filled the pass-throughconduit, the rotary valve is positioned so that the pass-through conduitis placed in fluid communication with both a liquid reagent and themixing chamber. Empirical studies have determined that a volume ofdiluent or liquid reagent or stain (referred to here as“diluent/reagent”) that is in excess of 3-times the volume of thepass-through conduit is sufficient to wash out the entire isolatedsample. In one example, the diluent/reagent is a combination of diluentand stain and is supplied in a volume to provide a dilution ratio ofabout 40:1. Therefore forty times the volume of the metering chamber canbe used to push the isolated sample out of the pass-through conduit andinto the mixing chamber, where the sample is uniformly mixed with thediluent/reagent, and then transferred into the imaging chamber.

The single use test cartridge contains an imaging chamber, through whichan automated microscope can acquire images of cells in the mixture ofdiluent/reagent and sample. In one embodiment of the present invention,the isolated sample is diluted with a known volume of diluent/reagent,whereby the dilution ratio is established. The volume of thediluent/reagent may be determined by measuring it, for example, bymonitoring its flow in a fluid channel of known dimensions with a cameraor other fluid sensor such as optical reflective/transmittive orultrasonic, or by temporarily metering it into a known volume chamberand then using only that known volume for sample dilution. According tothis embodiment, the cells in a known volume of the diluted sample arecounted, and because the dilution ratio is known, the cell counts perunit volume of the blood sample can be determined. The volume of thediluted sample can be known by filling an imaging chamber of knownvolume with the diluted sample. The volume of the imaging chamber may beknown by using highly reproducible manufacturing processes, or bymeasuring each test cartridge at time of manufacture and encoding sizingparameters in the package labeling. Alternatively, a measured amount ofthe diluted sample can be transferred into an imaging chamber of unknownvolume and counting all the cells in the chamber. A measured amount ofthe diluted sample can be determined by monitoring its flow from themixing chamber into a fluid channel of known dimensions by a camera, andisolating a segment. The segment, in turn, is moved into the imagingchamber.

In a preferred embodiment, the entire isolated sample is transferred tothe mixing chamber and then to the imaging chamber. According to thisembodiment, neither the dilution ratio nor the volume of the diluentneeds to be known. The size of the imaging chamber is chosen to ensurethat the entire volume of the isolated sample and the diluent/reagentcan be contained within the imaging chamber, but the exact dimensions orvolume of the chamber do not need to be known. The depth of the imagingchamber should be small enough to prevent the cells from overlapping atthe chosen dilution ratio when the cells settle to the bottom. Thisdepth is preferably between about 10 μm and 200 μm. In one embodiment,the depth of the imaging chamber is 100 μm and the ratio of thediluent/reagent to the isolated sample is 40 to 1. The width of theimaging chamber is chosen to provide uniform filling by the differentcell sizes and smooth flow without forming voids or crowding of cells.The length of the imaging chamber is calculated based on the depth andwidth parameters to provide the volume needed to accommodate the entireisolated sample at the chosen dilution ratio, and to provide a margin ofsafety. The shape of the imaging chamber may further be chosen to matchthe field of view of the digital camera or to facilitate capture ofmultiple images or to maintain a uniform distribution of cells

We have found that if the shape of the imaging chamber is square orrectangular having a ratio of length to width of 2:1, the cells will notuniformly fill the imaging chamber. If the mixture of sample anddiluent/reagent is uniform when it enters the imaging chamber, the cellswill tend to bunch and crowd, particularly near the sides or edges, andwhen they settle, the layer on the bottom of the imaging chamber willnot be homogenous. It is important to obtain a substantially homogenousmonolayer of cells in the imaging chamber, as this facilitates thecounting of all the cells. We have found that if the width and depth ofthe imaging chamber are small compared to the length, the cells remainmore uniformly distributed. Desirably, the length-to-width ratio of theimaging chamber is greater than 10:1. In various embodiments of thepresent invention, the shape of an imaging chamber may be serpentine,helical, or castellated according to the form factor of the testcartridge. In all of these cases the width and depth are small comparedto the length so that the cells due not aggregate on the sides orcorners of the imaging chamber, and the layer of cells settling to thebottom of the imaging chamber is substantially homogenous. Those skilledin the art will recognize that other geometries for the imaging chamber,which maintain the distribution of cells when the mixed solution ofsample and diluent/reagent is transferred into the imaging chamber mayalso be utilized.

The design goal of any imaging chamber is to contain all the cells fromthe original metered chamber and the diluent/reagent in a uniform mannerand without significant cell overlap when the cells settle to the bottomof the imaging chamber. The dilution ratio combined with the depth ofthe imaging chamber can be chosen to minimize the overlapping. As anillustrative example, an imaging chamber may have a width between 0.5 mmand 2.5 mm, a depth from 10 to 200 μm, and the dilution ratio may befrom 10:1 to 100:1.

Materials, which contact the cells outside of the imaging chamber, maybe chosen to have surface properties to minimize cell adherence. Liquidreagents, which may include a surfactant or cell sphering agent tofacilitate cell analysis, may also advantageously minimize red cellsbeing lost during transfer or being overlapped in the imaging chamber.The volume of liquid reagent and the flow velocity may also be chosen toimprove the likelihood of transferring every cell from the metering ormixing chamber to the imaging chamber and insuring that the distributionof the cells remains uniform. Yeh-Chan Ahn describes the complexconvective diffusion phenomena that is created in a serpentinemicrochannel which has a varying curvature (See Investigation of laminardispersion with optical coherence tomography and optical Dopplertomography, Yeh-Chan Ahn, Woonggyu Jung, Jun Zhang, and Zhongping Chen,OPTICS EXPRESS 8164, Vol. 13, No. 20, 3 Oct. 2005). Particles, or in ourcase, cells which are in suspension and moving through such amicrochannel tend to segregate according to their size, density, channelshape and flow velocity. Secondary flow from the serpentine geometry cancreate vortices as the channel curves causing the cells to be re-mixedinto the center of the flow at certain fill velocities. This re-mixinghelps maintain a near uniform distribution of the cells as the fluidfills the microchannel. In one embodiment, we have found that aserpentine path that is 1.25 mm wide with turning inside diameter of1.25 mm and an outside diameter of 2.5 mm, a depth of 0.125 mm, a lengthof 500 mm, and an effective fill speed of about 2 μL/s insures that theflow of cells remains uniform.

It should be noted that the time to count every cell is directly relatedto the volume of the metered chamber, and that this creates a trade-offin overall performance. Manufacturing highly reproducible meteringchambers is challenging for very small volumes. Therefore while it maybe easier and more reproducible to manufacture a metering chamber with a5 μL volume than one with a 1 μL volume, the 5 uL volume of sample wouldpotentially take five times longer to analyze. Embodiments of thepresent invention can provide a solution to this dilemma. By ensuringthat the sample and diluent/reagent are well mixed and that a properdilution ratio is chosen, and by utilizing a serpentine shaped imagingchamber where the length is large compared to the width and depth, thepattern of cells across the imaging chamber is relatively uniform andhighly reproducible. Under these conditions, the layer of cells settlingto the bottom of the imaging chamber will be a substantially homogenousmonolayer. As a result, instead of imaging the entire layer and countingevery cell, one can take representative images or frames that arestatistically derived to accurately account for every cell. Thus, withinthe allowable error for the final result(s), every cell is included inthe analysis, whether by actual image or by statistical representation.As an illustrative example, the camera may take up to 20,000 images at20× in order to scan the entire imaging chamber. Alternatively, everytenth frame could be taken to obtain a statistical representation.Another option would be to divide the imaging chamber into segments andcount the cells in every other segment or every third segment and so on.Another alternative to decrease the imaging time would be to scan theentire imaging chamber at 10×/0.25 NA or 4×/0.1 NA to obtain the totalWBC and RBC total counts, and then take images at higher power (20×.4 NAor higher) to obtain the higher-resolution detail needed for countingplatelets, reticulocytes, and performing the WBC differential.

Embodiments of the present invention can achieve accurate results thatare not dependent on many factors that have impacted the accuracy ofresults in the prior art. For example, the volume of the diluent/reagentand the dilution ratio do not impact the results, so long as all thecells from the metered volume are transferred to the imaging chamber andpresented for analysis. Similarly the representativeness of a selectedsub-volume of the mixture of sample and reagent, and the homogeneity ofthe sub-volume, which directly affect the accuracy of results by priorart methods, need have no impact on embodiments the present invention.Most importantly, the depth and uniformity of the imaging chamber, whichhas been difficult or expensive to control in other prior art efforts,need not impact the accuracy of results according to embodiments of thepresent invention.

The present disclosure further describes a cell analyzer that is smalland easy to use. The cell analyzer accepts the test cartridge andcarries out all the steps of the cell analysis without further inputfrom the user. In one embodiment, the test cartridge contains alldiluents and reagents needed to perform a CBC analysis of the patientsample placed in it. In this embodiment, the cell analyzer has fluidhandling components including positive and negative pressure sourcesthat interface with the test cartridge when it is placed into theanalyzer. One or more connections are made between the analyzer and thecartridge to place these pressure sources into fluid communication withchannels in the cartridge to provide a motive force to move liquidswithin the cartridge. The cell analyzer also has a mechanical valvedriver for operating a valve in the test cartridge for controlling themovements of fluids in the test cartridge. When the test cartridge isplaced in the analyzer, the mechanical valve driver is connected to avalve indexer to provide means for operating the valve in the testcartridge. The cell analyzer includes a mechanism for release of thediluent/reagent that are stored on board the test cartridge. The cellanalyzer further includes fluid control logic to automatically controlmovement of the fluids in the test cartridge by activating the positiveand negative pressure or displacement sources and operating themechanical valve driver according to pre-programmed sequences. The cellanalyzer may include a process monitoring camera positioned to acquiredigital images of the movement of fluids in the cartridge. Informationfrom the process monitoring camera can be used to provide feedback forthe fluid control logic or for monitoring critical steps.

The present disclosure also provides devices and methods for managingand/or monitoring the sample collection and preparation process toensure accurate results. In applications where the present invention isused to perform a CBC analysis, if too much time lapses betweenobtaining the blood sample and completing sample dilution and stainingwithin the test cartridge, the blood may clot or the cells may settleresulting in erroneous results. In one embodiment of the presentinvention that mitigates this risk, the test cartridge is placed on theanalyzer in a slide-out tray which initiates process monitoring withinthe analyzer. The blood sample is added to the cartridge by dispensing afree-hanging drop, or by using a capillary tube that is inserted intothe test cartridge. Immediately after the sample has been added to thecartridge, the user presses “Run Sample” to initiate the test sequence.Because the cartridge is controlled by the analyzer, the time to collectthe sample is known and can be short enough to avoid the need foranticoagulant coating or mixing the blood sample.

In an alternate embodiment that allows the sample to be collectedseveral minutes before processing, an anticoagulant, such as K2 or K3EDTA, is provided. This may be achieved by coating the sample input cupwith anticoagulant, by use of a capillary tube coated with anticoagulantfor a finger-stick sample, or by sampling from an evacuated bloodcollection tube such as a Becton Dickenson Vacutainer® containinganticoagulant. To manage cell settling, the test cartridge according tothis embodiment has a timing indicator which is initiated when the inputport is opened or the blood sample is introduced. The timing indicatorcan be a color-changing chemical reaction, a time delayed thermalreaction, an analog or digital timer, or other means known in the art.When the user loads the test cartridge into the analyzer, the timingindicator is read and if needed, the sample is mixed before proceeding.If too much time has lapsed the sample can be rejected to avoidproducing erroneous results.

In yet another embodiment that facilitates remote sample collection, thetest cartridge is docked to a carrier system. The carrier system is ahandheld or portable device used to facilitate a portion, or all, of thesample preparation steps. After blood is added to the test cartridgeusing any of the above mentioned methods, the carrier system accordingto this embodiment draws the blood into the cartridge, meters thesample, performs quality checks, and completes the sample preparation topresent the cells for image analysis. The carrier system would theneither eject the test cartridge for the user to transfer to the imaginganalyzer, or the carrier system could be docked to the imaging analyzerfor an automated handoff. In this embodiment anticoagulant coatings arenot needed and there is no risk of cell settling because the criticalsteps are initiated as soon as the sample is placed in the testcartridge.

Combinations of the workflow devices and methods are also contemplatedin the present disclosure. By way of example, a simple carrier systemcould incorporate a digital timer and a mechanism able to meter thesample, but not perform quality checks or full sample preparation. Theseadditional steps would be done by the cell analyzer.

The cell analyzer contains an automated microscope including anobjective lens, focusing mechanism, bright-field and fluorescent lightsources, filters, a dichroic mirror and a digital camera. In someembodiments, the cell analyzer may further include an illuminationsource and photometric detector for measuring light transmission at oneor multiple wavelengths for measuring the concentration of an analyte inthe sample. For example, the test cartridge can include a photometricchamber which is in fluid communication with the input port by means ofa fluidic channel, and through which the sample can be transferred forphotometric analysis, such as a hemoglobin measurement.

By way of example, to measure hemoglobin, the cell analyzer may have anillumination source consisting of two light emitting diodes (LEDs)providing excitation at wavelengths of 502 nm and 880 nm. The light pathfor the LEDs and a photometric detector are approximately 0.030″diameter. The 502 nm LED may be selected for absorbance measurementbecause of an isobestic point of oxyhemoglobin (O2Hb) anddeoxyhemoglobin (RHb). Also at 502 nm, the slope of the O2Hb and RHbcurves are very low, resulting in minimal variation with varying oxygensaturation (sO2) levels. Furthermore, the carboxyhemoglobin (COHb) curveis also close to this isobestic point, resulting in very little effectfrom COHb. An 880 nm LED can measure the background scatter effectscaused by RBCs, WBCs, lipids, etc. This is particularly important whentaking measurements on whole blood. This measurement may also beperformed on lysed blood, with or without a reagent for converting thehemoglobin to a single form, such as reduced hemoglobin, methemoglobin,azidemethemoglobin or cyanomethemogloin. In the case of a conversion toa single form of hemoglobin, a different peak wavelength for absorptionmeasurement may be used, such as 540 nm or 555 nm.

Disclosed and contemplated aspects also include an example of a testcartridge that is not preloaded with reagents, and instead, is coupledto a reagent supply module contained on board the cell analyzer. Theuser loads the reagent supply module into the cell analyzer where it isutilized for multiple test cartridges. When the reagent supply module isexhausted or expires, the cell analyzer alerts the user and will notperform additional tests until the reagent supply module is exchanged.The reagent supply module includes a cradle for receiving the testcartridge, a vessel for holding a liquid diluent/reagent, a diluentdelivery pump in fluid communication with the vessel, and adiluent/reagent output port constructed to interface with the testcartridge when the cartridge is in the cradle. In one example the sizeof the vessel is of sufficient capacity to provide diluent/reagents todilute several samples (50-100) with a reagent to sample ratio of 10:1to about 250:1. The diluent/reagent supply module may include aself-priming mechanism for priming the liquid reagent and eliminatingair bubbles. The reagent supply module may further include a chamber forcollecting waste diluent/reagent from the priming process.

In another embodiment contemplated in the present disclosure, a smallmeasured volume of whole blood may be mixed manually with an unknownamount of diluent/reagent in a sample preparation device. For instance,the known volume of sample and unknown diluent/reagent may be put into asample tube and gently rocked back and forth a few times. The entiremixed volume is then transferred by using a transfer pipette or similardevice to a test cartridge having an imaging chamber. According toembodiments of the present invention, if all of the cells in the imagingchamber are counted (either directly or by statistical sampling), thenthe concentration of cells per unit volume can be determined, withoutone needing to know the volume of the diluent/reagent, the dilutionratio, or the volume of the mixed sample in the imaging chamber, or thevolume of the imaging chamber occupied by the mixture.

Diluent/reagents that are preloaded in the test cartridge, or areprovided in a separate sample preparation device, or by a supply moduleare in a ready-to-use format. For a CBC analysis, the reagents mayinclude a membrane-permeable dye, such as Acridine Orange todifferentially stain the DNA and RNA of cells in whole blood. Otherstains known to those skilled in the art, such as cyanine dyes, can alsobe used to stain the blood cells. In an alternative arrangement, thestain may be provided in a dry reagent form together with a diluent thatis mixed with the dry reagent as needed. Multiple stains can be includedin a combination reagent. In one embodiment, the reagents may include anantibody conjugated to a detectable label that targets specific cells orspecific antigens associated with cells. The detectable label may be adye, a fluorescent dye, quantum dot, colloidal metal such as gold,silver, or platinum or other detectable constructs known in the art. Thedetectable label can also be used to detect a cell-specific antibody,such as CD3, CD4, CD14, CD16, CD19, CD34, CD45, CD56, or any other ofthe enumerated Cluster of Differentiation markers. The detectable labelcan also be used to detect bacterial or parasitic pathogens, platelets,recirculating tumor cells, leukemic cells, stem cells or any combinationof these.

In other embodiments the liquid reagent may contain a surfactant such aspolysorbate or sodium dodecyl sulfate (SDS), an anticoagulant such asEDTA, and/or a sphering agent such a zwitterionic detergent to provideisovolumetric reshaping of the red blood cells to facilitate cell sizemeasurement and computation of the mean corpuscular volume (MCV).

Systems according to the invention can exhibit better quantitativeaccuracy than manual microscope analyses using a manual hemocytomer orsimilar device, which tend to be limited by variability in samplepreparation and limited counting statistics. In embodiments of thepresent invention, sample preparation is improved by removing criticaloperator fluid handling steps and by automation of all dilution steps.Because every cell and platelet is counted in the entire metered volumeof sample, any error in the sample dilution is irrelevant.

Systems according to the invention can also save time that wouldotherwise be allocated to manual hemocytometer slide preparation, setuptime, and microscope focusing, which can limit the number of bloodsamples that can be analyzed. Automation can greatly increase the rateof image acquisition and analysis, allowing for more cells to beanalyzed and counted. This can improve the counting statistics andoverall precision of the system.

Systems according to the invention can also extend the capabilities ofcell counting methods by enabling CBC point-of-care testing, i.e. nearpatient testing, to permit immediate clinical decisions to be made.Personnel having a relatively low skill level can operate the systems.The analyzers can be engineered to be inexpensively manufactured andeasily serviced, allowing them to be more readily deployed atpoint-of-care sites, such as at the patient's bedside, in physician'soffices, and at emergency sites.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. The drawings are not necessarilyto scale, emphasis instead being placed upon illustrating the principlesof the invention. In the following description, various embodiments ofthe present invention are described with reference to the followingdrawings, in which:

FIG. 1 is a perspective view of an illustrative test cartridge beingpositioned to collect a drop of blood from a patient's finger;

FIG. 2A is a perspective view of an illustrative test cartridge with acover shown in the open position to receive a biological sample;

FIG. 2B is a perspective view of the test cartridge shown in FIG. 2Awith the cover shown in the closed position ready for analysis;

FIG. 3 is a cut-away view of an illustrative cell analyzer showinginternal components with a test cartridge being inserted;

FIG. 4 is a plan view of an illustrative test cartridge of the type thatincludes reagents for conducting a test;

FIG. 5 is a plan view of an illustrative test cartridge of the type thatdoes not include reagents;

FIG. 6 is a perspective view of an illustrative reagent supply moduleshowing a test cartridge ready to be joined with the module;

FIG. 7A is a perspective bottom view of a metering chamber formed in theface of a valve stem of a rotary valve;

FIG. 7B is a side view of a valve stem with a metering chamber formed asa pass-through conduit;

FIG. 8A is a plan view of an illustrative test cartridge showing asample of whole blood deposited in the input port area;

FIG. 8B is a plan view of the test cartridge of FIG. 8A showing initialmovement of the sample and reagent with the rotary valve in the firstopen position;

FIG. 8C is a plan view of the test cartridge of FIG. 8B with the valvein the second open position;

FIG. 8D is a plan view of the test cartridge of FIG. 8C illustrating thesample and the reagent in the imaging chamber;

FIG. 8E is a plan view of the test cartridge of FIG. 8D illustrating thesample and most of the reagent positioned in the mixing chamber;

FIG. 8F is a plan view of the test cartridge of FIG. 8E illustrating allof the sample and the reagent positioned in the imaging chamber and thevalve in a final, closed position;

FIG. 9 is an side elevation view of a cross section of the passivemixing chamber taken through line 9-9′ of FIG. 8E;

FIG. 10 is a plan view of a test cartridge of an alternate testcartridge embodiment.

FIG. 11 is a flowchart illustrating the operation of the cell analyzer.

FIGS. 12A and 12B show both bright-field and fluorescent images of thesame cells that were collected according to the present invention.

DETAILED DESCRIPTION

FIG. 1 illustrates test cartridge 100 being positioned to collect a dropof blood 120 from a patient's finger. The test cartridge is held beneaththe hanging drop 120, so that it contacts the input port 130 of the testcartridge 100. The input port 130 comprises a recessed area or openingthat may be coated with an anticoagulant and have surface treatment orfeatures such as small columns to increase surface retention to collectand hold the blood sample. In an alternate embodiment, the blood sample120 may be collected intravenously and introduced to input port 130 by atransfer pipette or capillary tube. The transfer pipette or capillarymay contain an anticoagulant coating according to the desired workflow.The volume of blood or other biological sample placed in input port 130is sufficient to visually fill the recessed sample area, but isunmeasured.

FIG. 2A shows test cartridge 100 with closure 135 shown in the openposition to provide access to input port 130. Closure 135 is adapted toslide relative to the test cartridge 100 and may have detent or otherpositioning features that facilitate placing it in different positions.After the biological sample has been collected into input port 130,closure 135 may be moved to the position shown in FIG. 2B to cover theinput port 130. The closure 135 may be moved by the user prior toinserting it into the analyzer as shown in FIG. 3. Alternatively,closure 135 may be moved by an operation within the cell analyzer.Alternate embodiments of closure 135 may include graphics, identifyinginformation, or instructions to the user. While the closure 135 isillustrated as a sliding component, other means of closing the inputport 130 are contemplated including a cap that hinges upward, a smallsurface cover that swivels away from and returns to cover the input port130, or an adhesive component that sticks to the input port 130 or areasurrounding it. In all cases the closure 135 includes a vent or air pathto the input port to allow the blood sample to move into the testcartridge 100.

FIG. 3 is a cut-away view of an illustrative cell analyzer 200 with testcartridge 100 positioned so that the operator can introduce it into theanalyzer. From the outside of the cell analyzer 200, one can see thehousing 206, a user-interface screen 208, a printer 212, and a cartridgeloading door 217. When the cartridge loading door 217 is opened, thetest cartridge 100 can be placed on a cradle 220 of x-y stage 225,configured to receive test cartridge 100 from the user. The cradle 220provides mechanical alignment of the cartridge to facilitate connectionsthat are made between the analyzer and the cartridge. For example, amechanical presser foot 230 may be placed in contact with a flexiblesurface on the test cartridge to provide mechanical pressure ontopackaged, on-board reagents. Some embodiments of the cell analyzer 200may utilize a reagent supply module 470 as further described withreference to FIG. 6. Reagent supply module 470 may be installed on x-ystage 225 and has a receiving area 473 (see FIG. 6) to provide alignmentof the test cartridge 402 with the reagent module 470.

A valve driver 235 can be positioned to operate a rotary valve on thetest cartridge. A vacuum/pressure pump 240 supplies negative or positivepressure to a manifold 245, which interfaces with the test cartridge 100when it is placed in the cell analyzer as described below. The cellanalyzer 200 further includes system controller 250 to control movementof the fluids in the test cartridge by activating the vacuum/pressurepump 240, moving the mechanical presser foot 230, or operating the valvedriver 235 according to pre-programmed sequences. Monitoring camera 255,positioned to acquire digital images of the fluids in the cartridge,provides feedback for the system controller 250. Monitoring light source256 may be a ring illuminator that surrounds the lens of the monitoringcamera 255. Information from the monitoring camera 255 is used toprovide feedback for controlling movement of liquids, for positioningthe rotary valve, and for confirming critical steps.

Also shown in FIG. 3 are the components that comprise the automatedmicroscope of the cell analyzer 200. At the base of the analyzer,bright-field light source 260 provides illumination through the testcartridge to the objective lens 265, operatively coupled to focusingmechanism 267. At the top of the analyzer, fluorescent light source 270provides illumination through dichroic mirror 277 to provide fluorescentexcitation of the sample. At the rear of the analyzer, digital camera280 captures images of the test cartridge 100 and transmits them toimage processor/computer 290. In some embodiments, the cell analyzer mayfurther include a photometric light source 293 and photometric detector295 for measuring light transmission at one or multiple wavelengths in achamber in test cartridge 100, such as for measuring hemoglobin, as ismore fully explained below.

FIG. 4 shows an illustrative test cartridge 401 of the type thatincludes liquid reagents stored in a blister pack 417 for conducting atest. The test cartridge 401 has an input port 407 for receiving asample, a passive mixing chamber 405 for mixing the sample withdiluent/reagent, and an imaging chamber 403 for capturing images of thecells in the mixture of sample and diluent/reagent for analysis. In thisembodiment, photometric chamber 409 may be filled with whole blood tomake optical absorbance measurements to determine concentrations ofcertain analytes in the sample, such as hemoglobin. A rotary valve 415provides fluidic connections between various fluidic channels, vents,and ports, including sample driver port 411, vent 423 and mixture driverport 429 as will be described in FIGS. 8A-8F.

FIG. 5 shows an illustrative test cartridge 402 of the type that doesnot include on-board diluent/reagents. Many of the functional componentsare identical to those illustrated with reference to test cartridge 401,but instead of on-board diluent/reagents, test cartridge 402 has areagent input port 460 adapted to be connected to an external source ofdiluent/reagent. Test cartridge 402 may be used in embodiments in whichdiluent/reagents that are needed for an analysis may be too costly topackage individually or may require refrigerated storage. In such anembodiment, diluent/reagent may be provided from a source within cellanalyzer 200 or from a reagent supply module.

FIG. 6 shows an illustrative reagent supply module 470 positioned toreceive test cartridge 402. The reagent supply module 470 includes areceiving area 473 for docking the test cartridge 402, and contains avessel for holding the diluent/reagent, a reagent metering pump adaptedto pump the diluent/reagent, and a reagent output port 475. The reagentoutput port 475 is constructed with a suitable shape and/or elastomericmaterials to insure a liquid-tight connection to reagent input port 460on the test cartridge 402, when the test cartridge is docked to thereagent supply module 470. Reagent supply module 470 has an opening 477suitably sized to allow monitoring camera 255 (FIG. 3) to image therotary valve 415. Additionally a window 478 in the reagent supply module470 is constructed to align with the photometric chamber 409 in the testcartridge. Window 478 allows the photometric light source 293 andphotometric detector 295 (FIG. 3) to make optical absorbancemeasurements on the fluid within photometric chamber 409.

In one embodiment, the size of the vessel within reagent supply module470 is of sufficient capacity to provide diluent/reagents to diluteand/or stain from ten to about one-hundred samples with adiluent/reagent to sample ratio of 10:1 to about 250:1. The reagentsupply module 470 further can include a self-priming mechanism forpriming the liquid reagent and eliminating air bubbles. In such anembodiment, the reagent supply module 470 may include a chamber forcollecting waste reagent from the priming process. Once the testcartridge 402 is docked with the reagent supply module 470 the combinedpieces perform the same functions as test cartridge 401 except that thereagent supply module 470 replaces the blister pack 417. Inside cellanalyzer 200 the vacuum/pressure pump 240 makes connections throughmanifold 245 to sample driver port 411 and mixture driver port 429. Theinterfaces between the manifold 245 and these ports are constructed witha suitable shape and/or elastomeric material to ensure an airtightconnection so that system controller 250 can control movement of thefluids in the test cartridge (see FIG. 3). In such an embodiment thepresser foot 230 is not needed.

The only volume that is measured precisely is the metered volume of theoriginal biological sample. Various means for metering a small volume ofliquid are well known in the art. Two devices that are well suited forlow cost, single use applications according to the present invention areshown in FIG. 7A and FIG. 7B. FIG. 7A shows the face of a cylindricalvalve stem 485 of a rotary face valve. Metering chamber 483 is formed inthe face by highly precise manufacturing processes such as injectionmolding. The chamber 483 is narrow and tubular in shape and centered inthe face of the cylindrical stem 485. A slot 487 in the top of stem 485acts as a valve indexer to indicate the position of the valve stem 485.Also formed in the face of valve stem 485 is an auxiliary connector 421,which has a circular shape. When assembled into the rotary valve 415(FIGS. 4 and 5), metering chamber 483 is able to connect between portsin the valve which are 180 degrees apart, while auxiliary connector 421connects between other ports which are 60 degrees apart. As will beexplained with reference to FIG. 8A-8F, system controller 250 is able tocontrol movement of the fluids by rotating valve stem 485 and bypositioning the valve according to the valve indexer 487 according topreprogrammed sequences. Thus in a first position, the metering chamber483 can be connected to the input port 407 (FIG. 4 and FIG. 5) andfilled with the biological sample, and then by rotating valve stem 485,the volume contained within metering chamber 483 can be isolated andtransferred for analysis.

FIG. 7B is a side view of a valve stem 485′ with a metering chamberformed as a pass-through conduit 413 in the tapered seat of valve stem485′. Pass-through conduit 413 is able to connect with fluidic channelsin rotary valve 415 which are 180 degrees apart. Also shown in FIG. 7Bis auxiliary fluidic connector 421′, which provides connections toadjacent fluidic channels which are 60 degrees apart.

When assembled in the rotary valve 415 (FIG. 4 and FIG. 5) having atapered seat to receive valve stem 485′, pass-through conduit 413 can beconnected to input port 407 (FIG. 4 and FIG. 5), filled with thebiological sample, and then by rotating valve stem 485′, the volume ofsample contained within pass-through conduit 413 can be isolated andtransferred for analysis. FIG. 7B also shows auxiliary fluidic connector421′, which provides fluidic connections to adjacent fluidic channels onthe test cartridge according to the position of the valve indexer 487′.It will be appreciated that the rotary face valve of FIG. 7A and thetapered seat valve of FIG. 7B are alternate embodiments for isolatingsample and controlling fluidic paths. Therefore, in the descriptionsthat follow references to metering chamber 483 in a rotary face valvewill be equally applicable to pass-through conduit 413 in a tapered seatvalve.

Now turning our attention to FIGS. 8A through 8F, and with reference toFIG. 3, a sequence of operations will be illustrated that enable cellanalyzer 200 to perform automated microscopic cell analysis on abiological sample without skilled operator interactions. In FIG. 8A asample is shown deposited into input port 407, which is in fluidcommunication with rotary valve 415. As illustrated in FIG. 8A, the stem485 (FIG. 7A) of rotary valve 415 is in a first position wherein themetering chamber 483 (FIG. 7A) is aligned with the sample input port 407and the sample driver port 411. A vacuum, supplied by the analyzer tosample driver port 411, draws the sample from the input port 407 intothe metering chamber 483 and into the photometric chamber 409. When thephotometric chamber 409 has been filled with sample, the systemcontroller 250 (FIG. 3) collects absorbance data from the undilutedsample using the photometric light source 293 (FIG. 3) and photometricdetector 295 (FIG. 3). As will be understood by those skilled in theart, suitable choice of optical wavelengths and chamber geometry andanalysis of the light passing through the biological sample can be usedto determine concentrations of certain analytes in the sample such ashemoglobin.

By illustration and with reference to FIG. 8B, cartridge 401 is shownwith a diluent/reagent contained in a blister pack 417. When rotaryvalve 415 positioned such that the metering chamber 483 is aligned withthe input port 407 and photometric chamber 409, auxiliary connector 421provides a fluid communication path between the blister pack 417 andvent 423. When pressure is applied to the blister pack 417 by presserfoot 230 (FIG. 3), diluent/reagent is released and flushed throughauxiliary connector 421 thereby priming the channels and removing airbubbles through vent 423.

FIG. 8C shows rotary valve 415 turned counterclockwise 60 degrees to asecond position, which isolates a predetermined amount of sample in themetering chamber 483. In this second position the stem 485 of rotaryvalve 415 is positioned such that the metering chamber 483 is in fluidcommunication with blister pack 417 and the serpentine imaging chamber403.

In FIG. 8D, the rotary valve 415 is shown in the same position as inFIG. 8C but following operation of the presser foot 230 which appliespressure to the blister pack 417. As illustrated by the shaded area, thediluent/reagent from blister pack 417 and the isolated sample 493 fromthe metering chamber 483 are transferred into the imaging chamber 403. Aminimum volume of reagent of three times the volume of the pass-throughconduit 413 is needed to flush the entire sample from the rotary valve415. According to the analysis being conducted, a sufficient volume ofthe reagent is pushed through the rotary valve 415 to completely washout the isolated sample and to achieve the approximate dilution ratiodesired.

In FIG. 8E the rotary valve 415 is shown turned counterclockwise 120degrees from its previous position shown in FIG. 8D to its thirdposition, wherein auxiliary connector 421 is aligned with mixture driverport 429 and imaging chamber 403. Vacuum/pressure pump 240 of cellanalyzer 200 (FIG. 3) supplies pressure to mixture driver port 429 andpushes all of the mixture of sample and diluent/reagent from the imagingchamber 403 into passive mixing chamber 405. As the mixture enterspassive mixing chamber 405, air within the chamber is vented throughvent port 433. Once all of the mixture of sample and diluent/reagent hasbeen transferred to the passive mixing chamber 405, vacuum/pressure pump240 applies a controlled vacuum to mixture driver port 429 such that themixture is pulled back into the imaging chamber 403. A preprogrammedsequence of pushing the mixture into the passive mixing chamber 405 andpulling it back into the imaging chamber 403 is repeated to achieve afinal mixture 495 that is free from cell clumping and overlapping afterthe cells settle to the bottom of the imaging chamber 403. In the finalmovement of the mixture 495, it is positioned entirely within theimaging chamber 403 as illustrated in FIG. 8F. We have found that thatin most instances, pushing the sample and diluent/reagent into mixingchamber 405 and pulling it out is sufficient to provide a uniformmixture. Further, the mixture remains substantially uniform when it istransferred into serpentine imaging chamber 403. It should also be notedthat the mixing chamber 405 could be located at the beginning of theimaging chamber 403.

FIG. 8F illustrates the final step of the sample preparation sequence.At this point in the preprogrammed sequence, the entire final mixture495 has been withdrawn from the passive mixing chamber 405 and ispositioned in the imaging chamber 403. When this position is achieved,the rotary valve 415 is rotated counterclockwise approximately 30degrees to the position shown in FIG. 8F, whereby it is not in fluidcommunication with any fluidic channel in rotary valve 415, therebyblocking further fluid communication with the imaging chamber 403 sothat no further movement of the final mixture 495 can take place.

FIG. 9 shows a cross section of the passive mixing chamber 405. Thechamber is referred to as “passive” because as illustrated, it does notcontain any active mixing element such as a bead or spin-bar. Suchdevices may be used in some embodiments, but we have found that anadequately sized chamber as depicted in FIG. 9 is simpler and providesexcellent mixing of the sample and reagent. In operation thediluent/reagent and sample 493 are driven by vacuum/pressure pump 240(FIG. 3) and enter and exit the chamber through mixing chamber opening497. As liquid enters the chamber, air within the chamber escapesthrough vent port 433. The cross section of passive mixing chamber 405illustrates wall geometry that increases smoothly in size from thebottom to the top such that the mixture entering from below expands intoa larger volume. The chamber 405 may have asymmetrical sloped walls 484and 491 to promote mixing of the sample and reagent and for removingbubbles from the mixture. After all of the mixture is in the chamber,air bubbles may be introduced to the chamber by vacuum/pressure pump 240through mixing chamber opening 497. These air bubbles further promotemixing and subsequently escape through vent port 433. The choice ofmaterials used to fabricate the passive mixing chamber 405 should takeinto consideration the wetting properties of the specific 1diluent/reagent(s) being utilized in the test cartridge 401. Theproperties of the material, among other requirements, should ensure thatliquid surface tension will pull back all of the liquid in contact withthe side walls of the chamber when the vacuum/pressure pump 240 emptiesthe chamber through mixing chamber opening 497.

FIG. 10 illustrates test cartridge 400 which comprises an imagingchamber 403 having at one end a sample input port 450, and at theopposite end a vent 453. A user of test cartridge 400 collects a smallknown volume of whole blood and mixes it manually with a diluent/reagentin a separate single-use sample preparation device (not shown). Oncemixed, the entire mixed volume is injected into sample input port 450.Air escapes through vent 453 as the mixture is injected allowing thesample to fill the imaging chamber 403. The imaging chamber isessentially the same imaging chamber as described above and shown inFIGS. 8A-8F. Test cartridge 400 can be placed into analyzer 200 (FIG. 3)for analysis beginning at step 560 of FIG. 11 as described below.

Turning our attention to FIG. 11 we will now describe the overalloperation of cell analyzer 200 configured to provide a “CBC withDifferential” analysis with reference to the test cartridge 401illustrated in FIGS. 8A-8F and cell analyzer 200 illustrated in FIG. 3.To obtain the blood sample from a patient presented at box 500, the userfirst obtains a new test cartridge 401 at box 505 and opens it to exposethe input port 407. Blood from a finger prick is applied as illustratedin FIG. 1 at box 510 and the input port 407 is covered. The user insertsthe test cartridge into the cell analyzer 200 at box 515. The testcartridge is moved into the analyzer where mechanical and fluidconnections are made between the analyzer and the cartridge as describedabove with reference to FIG. 3. As a first step of analysis, the sampleis drawn into the metering chamber passing through and into photometricchamber 409 (FIG. 8A). Absorbance of the blood is measured at box 520.Data from absorbance measurements are used to determine hemoglobinconcentration. At box 530 sample in the metering chamber 483 is imagedusing monitoring camera 255 and analyzed to confirm that the meteringchamber was properly filled at box 535. If an error is detected theanalysis is terminated at box 537 and the user is alerted to the errorand instructed to remove the cartridge and reject the test.

If the pass-through conduit 413 is correctly filled the diluent/reagentchannel is primed at box 540 as described above with reference to FIG.8B. Rotary valve 415 is then turned to the position shown in FIG. 8C toisolate the sample and to allow diluent/reagent to wash the meteredvolume of blood out of the pass-through conduit 413 at box 545 whilebeing imaged by monitoring camera 255. The transfer continues until themonitoring camera 255 confirms that diluent/reagent plus sample hasalmost filled the imaging chamber as illustrated in FIG. 8D.

Once a sufficient volume of diluent/reagent is transferred, rotary valve415 is positioned as shown in FIG. 8E and the total volume of sample anddiluent/reagent is mixed at box 550. At box 555 the entire volume 495 istransferred to the imaging chamber and rotary valve 415 is positioned asshown in FIG. 8F. Note that by transferring the entire volume of mixedsample 495, all of the metered volume of blood from the original sampleplus the unmetered volume of diluent/reagent is positioned in theimaging chamber at box 555.

If test cartridge 400 is used, it is inserted into cell analyzer 200 andanalysis begins at step 560. Analysis of test cartridge 401 or 402continues at step 560 when the x-y stage 225 moves the test cartridge401 to obtain bright-field and fluorescent images of the entire imagingchamber 403 at box 560. In an alternate embodiment, objective lens 265and/or digital camera 280 are moved and test cartridge 401 remainsstationary. In yet another embodiment objective lens 265 has sufficientfield of view to capture the entire imaging chamber 403 withoutmovement. Two digital images of each physical frame of the imagingchamber are transferred to image processor/computer 290 at box 565. Oneimage, taken with bright-field optics, can be compared to the otherimage taken with fluorescent optics to identify red blood cells, whiteblood cells and platelets. Further analysis of the white cell sizes andinternal structure can identify sub-types of white cells using patternrecognition.

At box 570 comparison of the bright-field and fluorescent images candifferentiate mature red cells from reticulocytes and nucleated redblood cells. By dividing each cell count by the known volume of themetering chamber 483, the concentration (cells per unit volume) can bedetermined. By using a sphering agent the planar sizes of red cells canbe transformed into mean corpuscular volume (MCV). Combining the redblood cell count with MCV and the volume of the metering chamber 483allows the calculation of hematocrit (HCT) and red cell distributionwidth (RDW). Further calculations using the separately measured HGB frombox 525, combined with the RBC count gives mean corpuscular hemoglobin(MCH), and mean corpuscular hemoglobin content (MCHC).

At box 575 the measured results are compared with previously definedlimits and ranges for the particular patient population anddetermination is made whether the results are within or outside normalexpected ranges. According to this determination results within normalranges are reported in box 580 and results that are outside the normalranges are reported in box 585.

EXAMPLES Example 1: Information from Bright-Field and Fluorescent Optics

FIG. 12 shows images that were collected using test devices according tothe present invention. A fluorescent stain Acridine Orange (AO) was usedto differentially stain DNA and RNA of cells in a whole blood sample.The visual images of FIG. 12 were obtained using an Olympus 20××0.4 NAobjective lens 265 and a Basler 5 MP digital camera 280. Excitation ofthe bright-field images in the second column was provided by white lightbright-field source 260. Excitation of the fluorescent images in thethird column was a 455 nm blue fluorescent light source 270.

White blood cells have significant RNA and DNA and therefore can be seenin the fluorescent images having green and orange structures. The sizeand shape of the green nuclear structure and overall size of the whitecells can be used to differentiate them into sub-groups identified byname in the first column. Notably the basophil and eosinophil sub-groupsof white cells have characteristic features in the bright-field imagedue to the presence of large granules in the cytoplasm. Thereforeembodiments of the present invention make use of both bright-field andfluorescent image analysis to differentiate sub-groups of white cells.

Platelets also take up the AO stain but the size of a platelet issignificantly smaller than any white cell and can therefore bedifferentiated. Because red cells lose their nucleus as they mature,they do not have nuclear material to take up the AO stain. Consequentlythe red cells can be identified as the objects that appear in thebright-field and cannot be seen in the fluorescent field. The immaturered cells, called reticulocytes and the nucleated red blood cells (nRBC)have attributes of red cells but also show small levels of fluorescence.Embodiments of the present invention make use of these combinedattributes to identify and sub-group red blood cells.

Example 2. Statistical Sampling of the Imaging Chamber

Table 1 illustrates a comparison of CBC parameters obtained according tothe present invention and from an automated hematology analyzer.

TABLE 1 Column 1 # pairs RBCs WBCs ROI RBC/f RBC/f(%)- WBC/f WBC/f(%)RBC/WBC 100% 9916 2455492 5125 3818.7 643.02 100.0 1.342 100.0 479.12 50% 4958 1229669 2535 1913.7 642.56 99.9 1.325 98.7 485.08  25% 2479623048 1285 968.5 643.28 100.0 1.327 98.9 484.86  10% 992 242197 519373.5 648.48 100.8 1.390 103.5 466.66  5% 496 126186 262 197.2 639.8299.5 1.325 99.0 481.63  1% 100 23683 63 35.6 664.61 103.4 1.768 131.7375.92Sample: Low WBC count—approximately 2000/uL (normal is 3,000-10,000/uL).Magnification: 20×Number of images: approximately 10,000 bright-field and 10,000fluorescentVariable: Column 1—Percentage of total cells use in the calculationColumn # pairs—the number of pairs of images (bright-field plusfluorescent)Column RBCs—total number of Red Blood Cells countedColumn WBCs—total number of White Blood Cells countedColumn ROI—total Region of Interest. This is the ‘effective’ number ofimage frames occupied by actual sample. A frame totally filled withsample/cells is “1”. A partial frame (due to an edge or the curved endsof the serpentine shape), is a fraction of a frame (e.g. 0.567).Column RBC/f—Average number of Red Blood Cells per frame (Column RBCsdivided by Column 5 ROI).Column RBC/f(%)—This is the RBC/frame value at a particular samplingpercentage divided by the RBC/frame for the 100% sampling case (topline). This is an estimate of the accuracy of the particular samplingpercentage compared to counting 100% of the cells.Column WBC/f—Average number of White Blood Cells per frame (Column WBCsdivided by Column ROI).Column 9 WBC/f(%)—This is similar to Column 7 but estimates the accuracyof the sampling percentage for the White Blood Cells.Column RBC/WBC—This is the ratio of RBC/WBC for the particular samplingpercentage.Results: A small percentage of the total frames can provide accurateresults. As a smaller fraction of the total frames are counted, theaccuracy is maintained down to 1% for Red Blood Cells and down to 5% forWhite Blood Cells.Discussion: In these experiments, it took approximately one second tocapture an image pair. For this experiment, where almost 10,000 imagepairs were needed to capture 100% of the sample, this means that imageanalysis took 10,000 seconds or approximately 2.8 hours. The experimentshows that the uniformity of the distribution of cells across theimaging chamber was good enough to provide accurate results by countingcells in only 5% of the frames. The goal of “counting every cell” isachieved because the entire sample size (the Region of Interest ROI) ismeasured, but only 5% of the images need to be analyzed to get accurateresults. This reduces the image analysis time to approximately 8minutes. It is expected that advances in camera and computer processingtechnology will further reduce this time.

The present invention has now been described in connection with a numberof specific embodiments thereof. However, numerous modifications whichare contemplated as falling within the scope of the present inventionshould now be apparent to those skilled in the art. Therefore, it isintended that the scope of the present invention be limited only by thescope of the claims appended hereto. In addition, the order ofpresentation of the claims should not be construed to limit the scope ofany particular term in the claims.

What is claimed is:
 1. A method for counting and analyzing biologicalparticles in whole blood including cells and platelets utilizing a cellanalyzer, a test cartridge having an imaging chamber, and diluent and/orstain, the method comprising: a) introducing a sample of the whole bloodinto the test cartridge; b) separating a known amount of the sample froma remaining amount of the sample in the test cartridge; c) mixing theknown amount of sample in the test cartridge with an amount of thediluent that is sufficient to form a substantially uniform mixture ofsample and diluent; d) transferring the mixture into the imaging chamberin the test cartridge; e) capturing one or more digital images of themixture in the imaging chamber that are selected to be statisticallyrepresentative of the number and distribution of biological particles inthe imaging chamber; f) counting all of at least one type of biologicalparticle in the images using imaging processing software; and g)calculating the number of particles per-unit volume of the one type ofbiological particles in the sample.
 2. A method of claim 1 wherein awidth and depth of the imaging chamber are uniform and thelength-to-width ratio of the imaging chamber is greater than 2 to
 1. 3.A method of claim 1 wherein a width of the imaging chamber is uniformand between 0.5 mm and 2.5 mm.
 4. A method of claim 1 wherein a depthand width of the imaging chamber are uniform and the width is 10 to 200um.
 5. A method of claim 1 wherein a ratio of diluent to sample in themixture of diluent and sample is between 10:1 to 250:1.
 6. A method ofclaim 1 wherein a depth of the imaging chamber is uniform and the shapeof the imaging chamber in planar view is serpentine.
 7. A method ofclaim 1 wherein a depth of the imaging chamber is uniform and the shapeof the imaging chamber in planar view is serpentine and having a widthof 1.25 mm, an inside turning radius of 1.25 mm, an outside turningradius of 2.5 mm, and a depth of 0.125 mm.
 8. A method of claim 7wherein the outside turning radius of the serpentine imaging chamber isabout twice the inside turning radius of the serpentine imaging chamber.9. A method of claim 1 wherein a rate of transferring of the mixture ofdiluent and sample into the imaging chamber is about 2 uL per second.10. A method of claim 1 wherein a shape of the imaging chamber in planarview is helical.
 11. A method of claim 1 wherein a shape of the imagingchamber in planar view is castellated.
 12. A method of claim 1 whereinthe particles settle to the bottom of the imaging chamber and wherein ageometry of the imaging chamber is such that the cells do not overlap,crowd, or stream when the particles settle to the bottom of the imagingchamber.
 13. A method of claim 1 further including the step of allowingthe particles in the mixture to settle to a bottom of the imagingchamber after the step of transferring the mixture into the imagingchamber.
 14. A method of claim 1 further comprising displaying thedigital images of the particles.
 15. A method of claim 1 furthercomprising analyzing the biological particles in the captured imageswith pattern recognition software.
 16. A method of claim 15 furthercomprising displaying the results of the analyzing the biologicalparticles.
 17. A method of claim 15 further comprising performing a fivepart differential of white cells.
 18. A method of claim 15 furthercomprising performing a three part differential of white cells.
 19. Amethod of claim 1 wherein the capturing of digital images includesbright field and fluorescent images.
 20. A method of claim 1 wherein thestep of capturing the digital images includes capturing images thatinclude all the particles in the imaging chamber.
 21. A method of claim1 wherein the known amount of sample is 0.1 uL to 5 uL and the amount ofdiluent is 1 uL to 500 uL.
 22. A method of claim 1 wherein a width anddepth of the imaging chamber are uniform and the length-to-width ratioof the imaging chamber is about 400 to
 1. 23. A method of claim 1wherein a geometry of the imaging chamber is such that a distribution ofparticles of the mixture remains substantially homogenous when themixture is transferred into the imaging chamber.
 24. A method of claim 1wherein the step of mixing includes mixing the sample with diluent andstain.
 25. A method of claim 24 wherein the step of mixing includes themixing of a stain in dry form.
 26. A method of claim 24 wherein the stepof mixing includes the mixing with a stain in liquid form.
 27. A methodof claim 24 wherein the known amount of sample is 0.1 uL to 5 uL and avolume of diluent and stain is 10 uL to 500 uL.
 28. A method of claim 24wherein the diluent and stain are in a same liquid before the step ofmixing.
 29. A method of claim 1 wherein the step of mixing includesmixing the sample with diluent and stain and cell sphering agent.
 30. Amethod of claim 1 wherein the steps of introducing, separating, mixing,transferring, and capturing are applied to a sample that includes atleast one of cells, platelets, sperm, bacteria, spores, and inorganicparticles.